Deciphering The Metabolic Signaling Pathways Regulated By Early Weaning Stress And Dietary Leucine Level In The Skeletal Muscle Of Piglets

Skeletal muscle is one of the main metabolic organs in the body,and the regulation of skeletal muscle is the key step for the whole metabolism under different physiological conditions.Early weaning is often one of the most stressful events in piglets,while early weaning stress and the nutritional strategies to support the growth of weaned piglets are the key issue in swine production.However,how early weaning stress alters the muscle protein expression and metabolic pathways in skeletal muscle of piglets,what metabolic roles for leucine in the regulation of skeletal muscle during post-weaning time are all poorly understood.Comparative proteomic analysis reveals the proteome profile alterations in an unbiased manner,which creating the opportunity to interpret metabolic signaling pathways regulated by early weaning stress and leucine in the skeletal muscle of piglets in tissue or cellular level.Thus,we have conducted the experiments as follow:Experiment 1:We applied isobaric tags for relative and absolute quantitation?iTRAQ?to analyze the proteome profile of the longissimus dorsi muscles of the piglets weaned for 0 h and 24 h.The main results are as follow:1.In this study,a total of 44 proteins were differentially expressed after 24 h early weaning stress.The differentially expressed proteins were characterized with bioinformatics tools,and those proteins have been enriched in the interaction networks.2.Early weaning stress alters the protein synthesis in the skeletal muscle of piglets.Pathway analysis showed that differentially expressed proteins are involved in several crucial protein synthesis signaling pathways,including EIF2 and mTOR signaling pathways.Western blot analysis showed that RPL17,RPS4 X,and p-mTOR/mTOR were significantly decreased?P<0?05?,these results suggested that early weaning stress inhibits muscle protein synthesis in the skeletal muscle of piglets.3.Early weaning with 24 h treatment significantly increased the protein level of LC3-II and the accumulation of autophagic vacuoles?P<0.01?,while PRKAR1 A,nuclear localization of p-CREB,ATG5,ATG12,and CAPNS1 were significantly increased in skeletal muscle after early weaning?P<0.05,except PRKAR1 A,P<0.01?.Our results suggest that early weaning stress up-regulates autophagy may mainly depends on PKA/CREB signaling pathways in the skeletal muscle of piglets.Experiment 2:We applied iTRAQ to analyze the proteome profile of the longissimus dorsi muscles of the weanling piglets fed with normal leucine diet?1.66%leucine,NL?and high leucine diet?2.1%leucine,HL?.The main results are as follow:1.157 differentially expressed proteins were identified between these two groups.Bioinformatics analysis of these proteins reveals leucine alters energy metabolism in the skeletal muscle.To further confirm,we detected SDHB,ATP5F1,ACADM,and HADHB were significantly down-regulated?P<0.01,except ATP5F1,P<0.05?,whereas glycolytic enzyme PKM was significantly up-regulated?P<0.05?in HL group.Simultaneously,the content of ATP in the skeletal muscle was also decreased?P<0.05?,all these results exhibited the suppression of oxidative phosphorylation and fatty acid?-oxidation,as well as the activation of glycolysis in HL group.2.Western blot analysis showed p-mTOR/mTOR,p-p70^S6K/p70^S6K,and RPS4 X were all up-regulated in HL group?P<0.05?,which suggested that leucine promotes muscle protein synthesis in weanling piglets.3.Leucine alters fiber type consititution in the skeletal muscle of piglets.We detected the TNNI1,MyHC-I,MyHC-IIa,and MyHC-IIx were all decreased?P<0.05,except TNNI1,P<0.01?,while TNNI2?P<0.01?and MyHC-IIb?P<0.05?were significantly increased in HL group.These results indicate that leucine may promote slow-to-fast transitions in the skeletal muscle of weanling piglets.